Phosphate-binding chitosan and uses thereof

ABSTRACT

The present invention provides compositions and methods for removing phosphate from a subject using chitosan. The present invention also provides compositions and methods for treating hyperphosphatemia based on phosphate-binding chitosan.

BACKGROUND OF THE INVENTION

Like other diseases for which there is no cure, chronic kidney diseasetakes an ever-increasing toll on patients who have it. As the diseaseprogresses, the kidney becomes less efficient at removing various ionsfrom the blood. Among these ions is phosphate, which can form insolubleparticles when combined with calcium. In end-stage renal disease, thefinal stage of chronic kidney disease, kidney function is so compromisedthat phosphate levels in the blood (serum) become markedly elevated.This condition, known as hyperphosphatemia, carries with it many gravehealth risks. For example, when serum phosphate and calcium levels areabove a certain threshold, hardened deposits may form throughout thebody, endangering circulation. It is therefore very important to controlserum phosphate levels in patients with end-stage renal disease.

Patients with end-stage renal disease may be advised to eat a diet lowin phosphate. However, phosphate is present at some level in almost allthe foods we eat. For this reason, phosphate binders were developed.Phosphate binders are compounds taken orally and which act in thegastrointestinal tract to bind phosphate and keep it from beingabsorbed. Phosphate binders are generally taken with each meal.Phosphate binders known in the art include, for example, various saltsof aluminum and calcium, as well as some chemically synthesizedcrosslinked polymers. There are clinical circumstances in which theadministration of aluminum or calcium salts is ill-advised. In animalmodels, certain crosslinked polymers carry with them elevated risks ofcarcinogenesis.

SUMMARY OF THE INVENTION

The present invention provides a safe and effective phosphate binderderived from a natural polymer. In particular, the present inventionprovides, inter alia, phosphate-binding chitosan, compositionscontaining phosphate-binding chitosan, and methods for treatinghyperphosphatemia using chitosan.

In one aspect, the present invention provides a method for removingphosphate from a subject (e.g., a mammalian subject). The methodincludes administering to the subject a therapeutically effective amountof chitosan. In some embodiments, the therapeutically effective amountof chitosan is administered orally.

In some embodiments, the subject is in need of treatment forhyperphosphatemia. In some embodiments, the subject is in need oftreatment for chronic kidney disease and/or end-stage renal disease. Insome embodiments, the subject is in need of treatment for one or moredisorders of phosphate metabolism and/or impaired phosphate transportfunction.

In some embodiments, the therapeutically effective amount ranges fromabout 0.1 to about 10 grams of chitosan per dose. In some embodiments,the therapeutically effective amount ranges from about 0.5 to about 50grams of chitosan per day.

In some embodiments, chitosan suitable for the invention binds at leastabout 30 mg phosphate per gram. In some embodiments, the chitosan bindsat least about 60 mg phosphate per gram. In some embodiments, thechitosan binds at least about 90 mg phosphate per gram. In someembodiments, the chitosan binds at least about 120 mg phosphate pergram. In some embodiments, the chitosan binds at least about 150 mgphosphate per gram. In some embodiments, the chitosan binds at leastabout 180 mg phosphate per gram.

In some embodiments, chitosan suitable for the invention is administeredin a form of a plurality of particles. In some embodiments, theplurality of particles have a mean volume particle size less than about100 cubic microns. In some embodiments, the plurality of particles havea median volume particle size less than about 100 cubic microns.

In some embodiments, the plurality of particles include one or moreparticles having a roundness greater than about 10. In some embodiments,at least about 0.3% of the plurality of particles have a roundnessgreater than 10. As used herein, “roundness” refers to a measurementdescribing the shape of a particle. As used in this application,roundness is defined by the following equation:

Roundness=(Perimeter²)/(4*pi*area)

Roundness is typically measured using a digital image of a population ofspheres and Image Pro Plus. Circular objects have a roundness=1.

In another aspect, the present invention provides a method for treatinghyperphosphatemia. The method includes administering to a subject inneed of treatment for hyperphosphatemia a composition comprisingchitosan. In some embodiments, the subject is in need of treatment forchronic kidney disease and/or end-stage renal disease. In someembodiments, the subject is in need of treatment for one or moredisorders of phosphate metabolism and/or impaired phosphate transportfunction.

In some embodiments, the composition of the invention includes atherapeutically effective amount of chitosan. In some embodiments, thetherapeutically effective amount is from about 0.1 to about 10 gramschitosan per dose. In some embodiments, the therapeutically effectiveamount is from about 0.5 to about 50 grams chitosan per day.

In some embodiments, chitosan suitable for the invention binds at leastabout 30 mg phosphate per gram. In some embodiments, the chitosan bindsat least about 60 mg phosphate per gram. In some embodiments, thechitosan binds at least about 90 mg phosphate per gram. In someembodiments, the chitosan binds at least about 120 mg phosphate pergram. In some embodiments, the chitosan binds at least about 150 mgphosphate per gram. In some embodiments, the chitosan binds at leastabout 180 mg phosphate per gram.

In some embodiments, chitosan suitable for the invention is present in aform of a plurality of particles. In some embodiments, the plurality ofparticles have a mean volume particle size less than about 100 cubicmicrons. In some embodiments, the plurality of particles have a medianvolume particle size less than about 100 cubic microns. In someembodiments, the plurality of particles include one or more particleshaving a roundness greater than about 10. In some embodiments, at leastabout 0.3% of the plurality of particles have a roundness greater than10.

In some embodiments, the composition suitable for the invention isadministered orally. In some embodiments, the composition is anutritional supplement. In some embodiments, the composition isadministered three times daily with meals.

In some embodiments, the composition further includes a carrier. In someembodiments, the carrier suitable for the invention is selected from thegroup consisting of a starch, a gum, an alginate, a silicate, dextrose,gelatin, lactose, mannitol, sorbitol, sucrose, tragacanth, cellulose,methyl cellulose, microcrystalline cellulose, a methylhydroxybenzoate, apropylhydroxybenzoate, polyvinylpyrrolidone and talc. In someembodiments, the composition is in a form of a cachet, a hard gelatincapsule, a soft gelatin capsule, an elixir, a lozenge, a pill, a powder,a sachet, a sterile packaged powder, a suspension, a syrup, or a tablet.

In yet another aspect, the present invention provides a compositionsuitable for treating hyperphosphatemia containing a therapeuticallyeffective amount of chitosan. In some embodiments, the hyperphosphatemiais associated with chronic kidney disease and/or end-stage renaldisease. In some embodiments, the hyperphosphatemia is associated withone or more disorders of phosphate metabolism and/or impaired phosphatetransport function.

In some embodiments, the therapeutically effective amount is from about0.1 to about 10 grams chitosan per dose. In some embodiments, thetherapeutically effective amount is from about 0.5 to about 50 gramschitosan per day.

In some embodiments, chitosan suitable for the invention binds at leastabout 30 mg phosphate per gram. In some embodiments, the chitosan bindsat least about 60 mg phosphate per gram. In some embodiments, thechitosan binds at least about 90 mg phosphate per gram. In someembodiments, the chitosan binds at least about 120 mg phosphate pergram. In some embodiments, the chitosan binds at least about 150 mgphosphate per gram. In some embodiments, the chitosan binds at leastabout 180 mg phosphate per gram.

In some embodiments, chitosan suitable for the invention is present in aform of a plurality of particles. In some embodiments, the plurality ofparticles have a mean volume particle size less than about 100 cubicmicrons. In some embodiments, the plurality of particles have a medianvolume particle size less than about 100 cubic microns.

In some embodiments, the plurality of particles include one or moreparticles having a roundness greater than about 10. In some embodiments,at least about 0.3% of the plurality of particles have a roundnessgreater than 10.

In some embodiments, the composition is suitable for oraladministration. In some embodiments, the composition is a nutritionalsupplement.

In some embodiments, the composition further includes a carrier. In someembodiments, the carrier is selected from the group consisting of astarch, a gum, an alginate, a silicate, dextrose, gelatin, lactose,mannitol, sorbitol, sucrose, tragacanth, cellulose, methyl cellulose,microcrystalline cellulose, a methylhydroxybenzoate, apropylhydroxybenzoate, polyvinylpyrrolidone and talc. In someembodiments, the composition is in a form of a cachet, a hard gelatincapsule, a soft gelatin capsule, an elixir, a lozenge, a pill, a powder,a sachet, a sterile packaged powder, a suspension, a syrup, or a tablet.

In still another aspect, the present invention provides a nutritionalsupplement containing chitosan, wherein the chitosan is present in aform of a plurality of particles having a mean volume particle size lessthan about 100 cubic microns.

In some embodiments, the plurality of particles include one or moreparticles having a roundness greater than about 10. In some embodiments,at least 0.3% of the plurality of particles have a roundness greaterthan about 10.

In a further aspect, the present invention provides a nutritionalsupplement containing chitosan, wherein the chitosan is present in aform of a plurality of particles having a median volume particle sizeless than about 100 cubic microns. In some embodiments, the plurality ofparticles have a mean volume particle size less than about 100 cubicmicrons.

In some embodiments, the plurality of particles include one or moreparticles having a roundness greater than about 10. In some embodiments,at least 0.3% of the plurality of particles have a roundness greaterthan about 10.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description, drawings and claims that follow.It should be understood, however, that the detailed description, thedrawings, and the claims, while indicating embodiments of the presentinvention, are given by way of illustration only, not limitation.Various changes and modifications within the scope of the invention willbecome apparent to those skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for removingphosphate from a subject using chitosan. The present invention alsoprovides compositions and methods for treating hyperphosphatemia using atherapeutically effective amount of chitosan.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Phosphate-Binding Chitosan

Chitosan suitable for the invention is a linear polysaccharide composedof β-(1-4)-linked D-glucosamine (deacetylated unit) andN-acetyl-D-glucosamine (acetylated unit). Chitosan typically has amolecular weight of approximately 10⁴ to 10⁶ dalton or higher. Chitosanis also referred to as poly-D-glucosamine; poly-[1-4]-β-D-glucosamine ordeacetylated chitin.

Typically, chitosan is derived from chitin (C ₈ H ₁₃ O ₅ N)_(n), along-chain polymer of a N-acetylglucosamine (a derivative of glucose)isolated from natural sources (e.g., cell walls of fungi, theexoskeletons of arthropods) by complete or partial deacetylation andpartial depolymerization. The deacetylation of chitin to chitosan can beperformed in hot concentrated NaOH solution (40-50%). Chitosan is alsocommercially available, for example, from Pronova Biopolymer, Inc.(Portsmouth, N.H.), e.g., as SEACURE 142, 242 or 342; from Vanson, Inc.(Redmond, Wash.) under the tradenames “Chitosan;” and from PrimexIngredients SA (Avaldsnes, Norway) under the tradenames “Chitoclear.”Other chitosan suppliers include, but are not limited to, AcroyaliHoldings Qingdao Co., Ltd. (Qingdao, China); AIDP, Inc. (City ofIndustry, CA); AK Biotech, Ltd. (Jinan, China); AK Scientific, Inc.(Mountain View, Calif.); Barrington Chemical Corporation (Harrison,N.Y.); Beckmann Chemikalien KG (Bassum, Germany); Carbomer, Inc. (SanDiego, Calif.); CCS CHEM. Co., Ltd. (Zhejiang, China); Dayang ChemicalsCo., Ltd. (Hangzhou, China); DNP International (Whittier, CA); DonbooAmino Acid Co., Ltd. (Jiangsu, China); EcoTag Comercial Ltd. (Cruz Alta,Brazil); Federal Laboratories Corporation (Alden, N.Y.); Fortune BridgeCo., Inc. (Elmont, N.Y.); Gallard-Schlesinger Industries, Inc.(Plainview, N.Y.); Hongkong Henry Industry Co., Ltd. (ZheJiang, China);Jiagen Biotechnologies, Inc. (Quebec, Canada); Jinan Haohua IndustryCo., Ltd. (Shandong, China); Kinbester Co., Ltd. (Xiamen, China);Kingreat Chemistry Co., Ltd. (Xiamen, China); Marcor DevelopmentCorporation (Carlstadt, N.J.); Marine Chemicals (Kerala, India); NantongChem-Tech. (Group) Co., Ltd. (Nantong, China); Ningbo Innopharmchem Co.,Ltd. (Ningbo, China); Ningbo Pangs Lanza International Co., Ltd.(Zhejiang, China); Nutriland Group Inc. (Torrance, Calif.); NutriScienceInnovations, LLC (Trumbull, Conn.); Orcas International, Inc. (Flanders,N.J.); Pacific Rainbow International, Inc. (City of Industry, Calif.);Panvo Organics Pvt. Ltd. (Tamil Nadu, India); Parchem Nutrition, Inc.(White Plains, N.Y.); Sears Phytochem Ltd. (Madhya Pradesh, India);SeaTech Bioproducts (Shrewsbury, MA); Shanghai Freemen Chemicals Co.,Ltd (Shanghai, China); Shanghai Mintchem Development Co., Ltd.(Shanghai, China); Shanghai Nicechem Co., Ltd. (Shanghai, China);Shanghai Sunwise Chemical Co., Ltd. (Shanghai, China); ShanghaiWellhoned Biotech Co., Ltd. (Shanghai, China); Sinosale Hebei Co., Ltd.(Shijiazhuang, China); Spectrum Chemicals & Laboratory Products(Gardena, Calif.); Stryka Botanics (Hillsborough, N.J.); VitajoyBio-tech Co., Ltd. (Suzhou, China); Wilke Resources, Inc. (Lenexa,Kans.); Wintersun Chemical (Ontario, Calif.); Wright Group (Crowley,La.); Xiamen Topusing Chemical Co., Ltd. (Xiamen, China).

Chitin, chitosan and chitin derivatives are further described inTharanathan et al., Crit. Reviews in Food Sci. & Nutrition, 43(1), pp.61-87 (2003), which is incorporated herein by reference.

Chitosan and chitin derivatives are often described according to thedegree of de-acetylation within the polysaccharide. Chitosan suitablefor the invention may have a range of degrees of de-acetylation. In someembodiments, chitosan suitable for the invention has a higher degree ofde-acetylation. In some embodiments, the degree of de-acetylation may beat least about 50% (e.g., at least about 60%, or at least about 70%, orat least about 80%, or at least about 85%, or at least about 87.5%, orat least about 90%, or at least about 92.5%, or at least about 95%, orat least about 97.5%, or at least about 98%, or at least about 99%, orat least about 99.5%, or at least about 99.9%). In some embodiments, thedegree of de-acetylation may range between 50% and 99.9% (e.g., between50% and 99%, or between 75% and 99.9%, or between 85% and 99.9%, orbetween 87.5% and 99.9%, or between 87.5% and 97.5%, or between 87.5%and 95%, or between 90% and 99.9%, or between 90% and 95%, or between95% and 99.9%, or between 95.5% and 97.5%, or between 97% and 99.9%, orbetween 98% and 99.9%, or between 98% and 99.5%, or between 99.5% and99.9%).

Chitosan suitable for the present invention also includes chitosanderivatives. Exemplary chitosan derivatives include, but are not limitedto, medium or long chain N-alkyl- or N-alkanoyl-chitosan, orwater-soluble chitosan. The term “medium chain N-alkyl- or N-alkanolyl”refers to C₈₋₁₃—N-alkyl- or —N-alkanolyl chains, the term “long chainN-alkyl- or N-alkanoyl” refers to C₁₄₋₁₈—N-alkyl- or —N-alkanolylchains. Examples of water-soluble chitosan include, but are not limitedto, CM-chitosan (carboxymethyl-chitosan), S-chitosan(oligosaccharide-chitosan), SCM-chitosan (N-sulfide derivative ofN-deacetylated CM chitin), HP-chitosan (hydroxyl-propyl-chitosan).

Suitable chitosan for the invention also includes any conventional saltsof chitosan. Examples of salts of chitosan include those with organicacids such as lower alkanoic acids, as well as mineral acids such as HCland H₂SO₄.

Suitable chitosan for the invention also includes any conventionalpharmaceutically acceptable acid of chitosan such as acetic, citric,formic and tartaric acid.

Suitable chitosan for the invention further includes modified chitosan.As used herein, “modified chitosan” refers to the chitosan obtained fromthe subsequent treatment of the initial product obtained from chitin.Exemplary modified chitosan includes, but is not limited to,semi-crystalline, microcrystalline, and nanoparticulate chitosan.Processes for obtaining modified chitosan are known in the art.Exemplary processes are described in U.S. Pat. Nos. 5,770,187,6,740,752, 6,638,918, 7,288,532, and PCT publications WO 01/32751, WO00/47177, the teachings of all of which are hereby incorporated byreference.

In some embodiments, chitosan suitable for the invention binds at leastabout 30 mg phosphate per gram chitosan. In some embodiments, chitosansuitable for the invention binds at least about 60 mg phosphate pergram. In some embodiments, chitosan suitable for the invention binds atleast about 90 mg phosphate per gram. In some embodiments, chitosansuitable for the invention binds at least about 120 mg phosphate pergram. In some embodiments, chitosan suitable for the invention binds atleast about 150 mg phosphate per gram. In some embodiments, chitosansuitable for the invention binds at least about 180 mg phosphate pergram chitosan. In some embodiments, chitosan suitable for the inventionbinds at least about 210 mg phosphate per gram. In some embodiments,chitosan suitable for the invention binds at least about 240 mgphosphate per gram.

Without wishing to be bound by any theories, it is contemplated thatchitosan bind and remove phosphate through an ion exchange process. Asused herein, the term “ion exchange” has its ordinary meaning in thechemical and/or pharmaceutical field. In particular, ion exchange can bea process including the release of one or more anions ionically bound toa cationic polymer and the subsequent ionic binding of another one ormore anions to the polymer. As a non-limiting example, ion exchangeincludes the release of one or more chloride ions from a polymer such aschitosan and the subsequent binding of one or more phosphate, hydrogenphosphate, and/or dihydrogen phosphate ions to the polymer.

Particles of Chitosan

In particular, chitosan suitable for the present invention may be in aform of particles. In some embodiments, chitosan suitable for theinvention are present in a form of a plurality of particles. Thechitosan particulates suitable for the invention can have a broadparticle size distribution. Typically, the chitosan particles can becharacterized by a mean volume particle size, and/or a median volumeparticle size. In some embodiments, the chitosan particles suitable forthe invention can have a mean volume particle size less than about 300cubic microns, or less than about 250 cubic microns, or less than 200cubic microns, or less than 150 cubic microns, or less than about 100cubic microns, or less than about 75 cubic microns, or less than about50 cubic microns. As used herein, 1 cubic micron equals to 1 μm³. Insome embodiments, the chitosan particles have a mean volume particlesize less than about 100 cubic microns.

In other embodiments, the chitosan particles suitable for the inventioncan have a median volume particle size less than about 300 cubicmicrons, or less than about 250 cubic microns, or less than 200 cubicmicrons, or less than 150 cubic microns, or less than about 100 cubicmicrons, or less than about 75 cubic microns, or less than about 50cubic microns. As used herein, 1 cubic micron equals to 1 μm³. In someembodiments, the chitosan particles have a median volume particle sizeless than about 100 cubic microns.

In some embodiments, the chitosan particles suitable for the inventionmay have combinations of median volume particle size and mean volumeparticle size as described above. For example, the chitosan particlessuitable for the invention may have a median volume particle size lessthan about 100 cubic microns and a mean volume particle size less thanabout 100 cubic microns.

A chitosan particle can also be characterized by a roundness. As usedherein, the term “roundness” has its ordinary meaning in the particlesize and shape measurement arts. As used in this application, roundnessis defined by the following equation:

Roundness=(Perimeter²)/(4*pi*area)

Roundness is typically measured using a digital image of a population ofspheres and Image Pro Plus. Circular objects have a roundness=1.

Typically, chitosan particles suitable for the invention can have arange of roundness. For example, a plurality of chitosan particlessuitable for the invention may have particles with a roundness greaterthan 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0,5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, or greater. In someembodiments, a plurality of chitosan particles suitable for theinvention include one or more particles having a roundness greater than10. In some embodiments, at least about 0.1% of the plurality ofchitosan particles used in a composition of the invention have aroundness greater than 10. In some embodiment, at least about 0.2% ofthe plurality of chitosan particles used in a composition of theinvention have a roundness greater than 10. In some embodiment, at leastabout 0.3% of the plurality of chitosan particles used in a compositionof the invention have a roundness greater than 10. In some embodiment,at least about 0.4% of the plurality of chitosan particles used in acomposition of the invention have a roundness greater than 10. In someembodiment, at least about 0.5% of the plurality of chitosan particlesused in a composition of the invention have a roundness greater than 10.In some embodiment, at least about 1.0% of the plurality of chitosanparticles used in a composition of the invention have a roundnessgreater than 10. In some embodiment, at least about 5.0% of theplurality of chitosan particles used in a composition of the inventionhave a roundness greater than 10.

In some embodiments, the chitosan particulates suitable for theinvention are cationic particulates. In some embodiments, the chitosanparticulates suitable for the invention can be anionic/cationicamphoteric particulates or anionic particulates. Processes for producingchitosan particulates are well known in the art. For example, materialsin which chitosan having a primary amino group as a base can bedissolved in an acid and the resultant solution can be dropped into analkaline coagulation fluid to produce cationic particulates. As anothernon-limiting example, chitosan solutions or dispersions can bemechanically treated to generate particulates.

Treatment of Hyperphosphatemia

Phosphate-binding chitosan as described above can be used to treathyperphosphatemia. As used herein, the term “hyperphosphatemia” refersto a higher than normal blood level of phosphorous. In human adults, thenormal range for blood phosphorous is approximately 2.5-4.5 mg/dL (i.e.,2.5-4.5 mg/100 ml). Typically, an individual with hyperphosphatemiacondition has fasting serum phosphorus concentration higher than 5.0mg/dL (e.g., higher than 5.5 mg/dL, 6.0 mg/dL, 6.5 mg/dL, or 7.0 mg/dL).Methods for measuring phosphate concentrations are well known in theart. For example, phosphate concentrations can be quantitated by themethod of Lowry and Lopez, J. Biol. Chem. 162: 421-428. Thehyperphosphatemia condition, especially if present over extended periodsof time, leads to severe abnormalities in calcium and phosphorusmetabolism and can be manifested by aberrant calcification in joints,lungs, and eyes.

Hyperphosphatemia is associated with various diseases or medicalconditions including, but not limited to, diseases associated withinadequate renal function such as, for example, chronic kidney diseaseand/or end-stage renal disease, hypoparathyroidism, and other disordersof phosphate metabolism and/or impaired phosphate transport function.

As used herein, an “individual,” “patient” or “subject” being treatedincludes a human or a non-human such as, a non-human mammalian subjectincluding, but not limited to, a bovine, cat, dog, ferret, gerbil, goat,guinea pig, hamster, horse, mouse, nonhuman primate, pig, rabbit, rat,or sheep.

The term, “treat” or “treatment,” as used herein, includes achieving atherapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication, amelioration, or prevention of theunderlying disorder being treated. For example, in a hyperphosphatemiapatient, therapeutic benefit includes eradication or amelioration of theunderlying hyperphosphatemia. Also, a therapeutic benefit is achievedwith the eradication, amelioration, or prevention of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Inparticular, treating hyperphosphatemia includes reducing or decreasingserum phosphate concentration. As used herein, the terms “reduce” or“decrease,” and grammatical equivalents, indicate values that arerelative to a baseline measurement, such as a measurement in the sameindividual prior to initiation of the treatment described herein, or ameasurement in a control individual (or multiple control individuals) inthe absence of the treatment described herein. A “control individual” isan individual afflicted with the same condition of hyperphosphatemia asthe individual being treated. For prophylactic benefit, the compositionof the invention may be administered to a patient at risk of developinghyperphosphatemia or to a patient reporting one or more of thephysiological symptoms of hyperphosphatemia, even though a diagnosis ofhyperphosphatemia may not have been made.

In general, a method of treating hyperphosphatemia includesadministering to a subject a therapeutically effective amount ofchitosan. As used herein, the term “therapeutically effective amount”refers to an amount effective to achieve therapeutic or prophylacticbenefit as described above. The therapeutically effective amount can beadministered in a single dose or in a series of doses separated byappropriate time intervals, such as hours or days. For example, atherapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. An appropriate unit dosewithin an effective dosing regimen is also referred to as“therapeutically effective dose.”

The actual amount effective for a particular application will depend onthe condition being treated (e.g., the disease or disorder and itsseverity, and the age and weight of the patient to be treated) and theroute of administration. Determination of an effective amount is wellwithin the capabilities of those skilled in the art, especially in lightof the disclosure herein. For example, the effective amount for use inhumans can be determined from animal models. For example, a dose forhumans can be formulated to achieve circulating and/or gastrointestinalconcentrations that have been found to be effective in animals. In someembodiments, a therapeutically effective amount of chitosan can be atleast about 0.1 grams per dose, or at least 2 grams per dose, at least 4grams per dose, or at least 6 grams per dose, or at least, 8 grams perdose, or at least 10 grams per does, or at least 15 grams per dose, orat least 20 grams per dose. In some embodiments, a therapeuticallyeffective amount of chitosan is from about 0.1 to about 10 grams perdose. In some embodiments, a therapeutically effective amount ofchitosan is from about 0.1 to about 20 grams per dose. In someembodiments, a therapeutically effective amount of chitosan is fromabout 0.1 to about 5 grams per dose. In some embodiments, thetherapeutically effective amount of chitosan can be at least about 0.5grams per day, or at least 5 grams per day, at least 10 grams per day,or at least 15 grams per day, or at least 20 grams per day, or at least25 grams per day, or at least 30 grams per day, or at least 35 grams perday, or at least 40 grams per day, or at least 45 grams per day, or atleast 50 grams per day, or at least 55 grams per day, or at least 60grams per day. In some embodiments, the therapeutically effective amountof chitosan ranges from about 0.5 to about 50 grams per day. In someembodiments, the therapeutically effective amount of chitosan is fromabout 0.5 to about 25 grams per day. In some embodiments, thetherapeutically effective amount of chitosan is from about 0.5 to about100 grams per day. In some embodiments, the therapeutically effectiveamount of chitosan is from about 10 to about 150 grams per day.

In some embodiments, the chitosan of the invention is used together withone or more other phosphate binders to treat hyperphosphatemia. Forexample, the chitosan may be used together with aluminium hydroxide(Alucaps®), calcium carbonate (Calcichew®, Titralac®), calcium acetate(Phosex®, PhosLo®), lanthanum carbonate (Fosrenol®), or sevelamer(Renagel®, Renvela®).

Compositions Containing Phosphate-Binding Chitosan

The chitosan used for therapeutic and/or prophylactic benefits can beadministered alone or in the form of a composition as described herein.A composition of the invention typically contains a therapeuticallyeffective amount (or a therapeutically effective dose) ofphosphate-binding chitosan as described above. Typically, the percentageof chitosan in a composition of the invention is at least about 0.005%by weight of the composition (e.g., at least about 0.1%, 0.5%, 1.0%,1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30% orhigher). In some embodiments, the percentage of chitosan in acomposition of the invention ranges from about 0.1% to about 30% basedon the weight of the composition. In some embodiments, the percentage ofchitosan in a composition of the invention ranges from about 0.1% toabout 10% based on the weight of the composition. In some embodiments,the percentage of chitosan in a composition of the invention ranges fromabout 0.2% to about 5% based on the weight of the composition. In someembodiments, the percentage of chitosan in a composition of theinvention ranges from about 0.35% to about 1.0% based on the weight ofthe composition.

Typically, a composition of the invention further includes a carrier. Acarrier suitable for the invention is also referred to as apharmaceutically acceptable carrier or a carrier-diluent. A carrier maybe a solid, semi-solid or liquid material which acts as an excipient,medium, and/or vehicle for chitosan. For example, a composition of theinvention can be in a solid or liquid medium. For example, chitosan maybe enclosed within a carrier, such as a capsule, paper, sachet or othercontainer. In particular, a suitable carrier, excipient, or diluent maybe a starch, a gum, an alginate, a silicate, dextrose, gelatin, lactose,mannitol, sorbitol, sucrose, tragacanth, cellulose, methyl cellulose,microcrystalline cellulose, a methylhydroxybenzoate, apropylhydroxybenzoate, polyvinylpyrrolidone or talc.

The chitosan can be administered by injection, topically, orally,transdermally, or rectally. The composition containing chitosan can beformulated to suit the mode of administration. In some embodiments, acomposition of the present invention is formulated for oraladministration. For example, a composition according to the inventionmay be in a form of a cachet, a hard gelatin capsule, a soft gelatincapsule, an elixir, a lozenge, a pill, a powder, a sachet, a sterilepackaged powder, a suspension, a syrup, a tablet, a capsule, solution,or emulsion, to name but a few.

In some embodiments, a composition of the invention can be a food, adrink, or a nutritional, food or dietary supplement. In one embodiment,the composition is a nutritional supplement. As used herein, “anutritional supplement” is a preparation formulated to supply nutrients(including, but not limited to, vitamins, minerals, fatty acids or aminoacids) that are missing or not consumed in sufficient quantity in aperson's or animal's diet. As used in this application, a nutritionalsupplement is also referred to as “a food supplement” or “a dietarysupplement.”

In some embodiments, the composition of the invention is a nutritionalsupplement for a person's diet. The nutritional supplement can beadministered with or without meals and can be administered once daily,twice daily, three times daily, once every other day, twice a week, oncea week, or at a variable intervals. In some embodiments, the nutritionalsupplements can be administered three times daily with meals.Supplements may be in various forms including, for example, powders,liquids, syrups, pills, encapsulated compositions, etc.

In some embodiments, the composition of the invention is a nutritionalsupplement for an animal's diet, such as, a feed or pet food used withanother feed or pet food to improve the nutritive balance or performanceof the total. Contemplated supplements include compositions that are fedundiluted as a supplement to other feeds or pet foods, offered adlibitum with other parts of an animal's ration that are separatelyavailable, or diluted and mixed with an animal's regular feed or petfood to produce a complete feed or pet food.

In another embodiment, a composition of the invention can be a treat foranimals. Treats include, for example, compositions that are given to ananimal to entice the animal to eat during a non-meal time. Contemplatedtreats for canines include, for example, dog bones. Treats may benutritional, wherein the composition comprises one or more nutrients,and may, for example, have a composition as described above for food.Non-nutritional treats encompass any other treats that are non-toxic.Chitosan can be coated onto the treat, incorporated into the treat, orboth.

Typically, the chitosan and other ingredients of the composition arepresent at concentrations that do not impart, when combined, an odor orflavor that causes the intended animal to perceive the composition to beunacceptable for consumption. In many instances, a desirable odor andflavor can be achieved using aroma or flavor enhancers.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1 Phosphate-Binding Assay

Phosphate concentrations were quantitated by the method of Lowry andLopez, J. Biol. Chem., 162: 421-428. To determine phosphate binding tochitosan powder, 20 mg chitosan was weighed into a 12×75 mm glass tube.3.0 ml of distilled water and 50 μl of 0.53M monobasic potassiumphosphate were added. The tube was sealed with plastic wrap and placedon a Lab-Tek Aliquot mixer to mix for 1 hour at room temperature. Thechitosan was then allowed to settle by gravity.

3.0 ml of acetate buffer (0.1N acetic acid, 0.025N sodium acetate) wasadded into a new clean, dry, and optically matched 12×75 mm glass tube.50 μl of supernatant was taken from the chitosan tube as prepared aboveand mixed into the acetate buffer. Next, 0.30 ml of 1% ascorbic acid wasadded with mixing followed by 0.30 ml of 1% ammonium molybdate in 0.05Nsulfuric acid with mixing. The absorbance at 700 nm was measured in aSpectronic 20 spectrophotometer 10 minutes after adding the molybdate todetermine the amount of phosphate remaining in the chitosan supernatant.A blank tube containing no chitosan and no phosphate and a phosphatestandard containing no chitosan were run through the entire assaysimultaneously as controls.

To calculate the amount of phosphate bound to chitosan, the absorbanceof the blank was subtracted from the absorbance of all the other samplesto obtain the corrected absorbance. The phosphate concentrationremaining in the chitosan supernatant was calculated on the basis of thecorrected absorbance of the phosphate standard compared to that of thechitosan sample. The total amount of phosphate remaining in the chitosansupernatant was then subtracted from the total amount of the initialphosphate to obtain the amount bound to chitosan and expressed as mgphosphate bound per gram of chitosan powder.

Exemplary phosphate-binding results are summarized in Table 1.

TABLE 1 Exemplary phosphate-binding activities Sample PO₄ binding, mg/gA 0 < y ≦ 30 B 0 < y ≦ 30 C 0 < y ≦ 30 D 0 < y ≦ 30 E 0 < y ≦ 30 F 30 <y ≦ 60  G 30 < y ≦ 60  H 60 < y ≦ 90  I 60 < y ≦ 90 

The degree of de-acetylation was also determined using standard methodsand exemplary results are summarized in Table 2.

TABLE 2 Exemplary results of de-acetylation degree Sample %De-acetylation A 87.5 < x ≦ 91.5 B 87.5 < x ≦ 91.5 C 87.5 < x ≦ 91.5 D87.5 < x ≦ 91.5 E 87.5 < x ≦ 91.5 F 91.5 < x ≦ 94.7 G 91.5 < x ≦ 94.7 H94.7 < x ≦ 97.1 I 94.7 < x ≦ 97.1

The exemplary degrees of de-acetylation shown in Table 2 arenon-limiting examples. Indeed, chitosan samples with a range of degreeof de-acetylation can be used to bind phosphate, including chitosansamples with de-acetylation degree lower than 87.5% (e.g., lower than85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or lower).

Without wishing to be bound by any theory, it is however contemplatedthat higher degree of de-acetylation may enhance phosphate-bindingactivity of chitosan. Chitosan samples with higher degrees ofde-acetylation can be prepared and the predicted phosphate-bindingactivities are shown in Table 3.

TABLE 3 Additional exemplary phosphate-binding activities Sample %Deacetylation (x) PO₄ binding (y), mg/g J 97.1 < x ≦ 98.7  90 < y ≦ 120K 98.7 < x ≦ 99.5 120 < y ≦ 150 L 99.5 < x ≦ 99.9 150 < y ≦ 180 M 99.9 <x 180 < y ≦ 210

Example 2 The Size and Shape of Chitosan Particles and TheirPhosphate-Binding Abilities

Certain samples of chitosan were subjected to particle size and shapeanalysis using techniques known in the particle size measurement art.For example, samples of chitosan particles can be analyzed using a Nikonmicroscope and Image-Pro Plus.

To analyze a sample using Image-Pro Plus, the sample was first dispersedinto a carrier fluid. It was then put under the microscope and themagnification was determined. In the Image-Pro Plus program, refiningadjustments were made to get an optimal image with good contrast so thatthe program can distinguish the particles from the background.Typically, 100 images were then taken at random to get an unbiasedresult. Typically, at least 300 particles were needed to count in the100 images.

Once the images were collected, a Macro was run on Image-Pro Plus tocount the particles and calculate the specific statistics as needed(e.g., roundness, area or size). For example, roundness was calculatedby the program using the formula (perimeter²)/(4*p*area). Circularobjects have a roundness=1.

The size distribution of a chitosan sample was typically characterizedby a mean volume particle size and/or a median volume particle size.

The phosphate-binding abilities of chitosan samples were determined asdescribed in Example 1.

A possible relationship between particle size and the presence orabsence of phosphate-binding activity was determined. Exemplary resultsare summarized in Table 4.

TABLE 4 Chitosan sample description Volume mean, μm³ Volume median, μm³Phosphate binder <100 <100 Phosphate non-binder alpha >100 >100Phosphate non-binder beta >100 >100

A possible relationship between particle shape (roundness) and thepresence or absence of phosphate-binding activity was also determined.Exemplary results are summarized in Table 5.

TABLE 5 Max. % particles with Chitosan sample description particleroundness roundness greater than 10 Phosphate binder >10 0.3% Phosphatenon-binder alpha <10 0 Phosphate non-binder beta <10 0

Example 3 Treatment of Hyperphosphatemia Using Chitosan

The following human patients suffering from hyperphosphatemia aretreated with chitosan as described below.

Human patient No. 1 has a serum phosphorus concentration between about5.5 and about 7.5 mg/dL and has not taken a phosphate binder prior tothe treatment. A composition containing about 4 grams of chitosan sampleI from Table 1 is orally administered three times daily with meals.

Human patient No. 2 has a serum phosphorus concentration between about7.5 and about 9.0 mg/dL and has not taken a phosphate binder prior tothe treatment. A composition containing about 6 grams of chitosan sampleI from Table 1 is orally administered three times daily with meals.

Human patient No. 3 has a serum phosphorus concentration greater thanabout 9.0 mg/dL and has not taken a phosphate binder prior to thetreatment. A composition containing about 8 grams of chitosan sample Ifrom Table 1 is orally administered three times daily with meals.

Human patient No. 4 suffers from hyperphosphatemia and has been takingone 667-mg calcium acetate tablet per meal. A composition containingabout 2 grams of chitosan sample K from Table 3 is orally administeredthree times daily with meals, instead of the one 667-mg calcium acetatetablet per meal.

Human patient No. 5 suffers from hyperphosphatemia and has been takingtwo 667-mg calcium acetate tablets per meal. A composition containingabout 3 grams of chitosan sample K from Table 3 is orally administeredthree times daily with meals, instead of the two 667-mg calcium acetatetablets per meal.

Human patient No. 6 suffers from hyperphosphatemia and has been takingthree 667-mg calcium acetate tablets per meal. A composition containingabout 5 grams of chitosan sample K from Table 3 is orally administeredthree times daily with meals, instead of the three 667-mg calciumacetate tablets per meal.

Human patient No. 7 has a serum phosphorus level between 5.5 and 6.0mg/dL and has not taken a phosphate binder prior to the treatment. Acomposition containing about 0.15 grams of chitosan sample M from Table3 is orally administered three times daily with meals.

In each of the cases, the patient's hyperphosphatemia is controlled. Insome cases, the patent's blood phosphate level is lowered after chitosantreatment. The dosing can be adjusted during treatment based on thepatient's condition and the blood phosphorus level. Typically, thedosing regimens are maintained unchanged when the human patients' serumphosphorus levels are reduced to and remain in the range from 3.5 to 5.5mg/dL, in which case the patient's hyperphosphatemia is beingcontrolled.

Equivalents

The foregoing has been a description of certain non-limiting embodimentsof the invention. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Those ofordinary skill in the art will appreciate that various changes andmodifications to this description may be made without departing from thespirit or scope of the present invention, as defined in the followingclaims.

In the claims articles such as “a,”, “an” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the claims or from relevant portions of thedescription are introduced into another claim. For example, any claimthat is dependent on another claim can be modified to include one ormore limitations found in any other claim that is dependent on the samebase claim. Furthermore, where the claims recite a composition, it is tobe understood that methods of using the composition for any of thepurposes disclosed herein are included, and methods of making thecomposition according to any of the methods of making disclosed hereinor other methods known in the art are included, unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise. In addition, theinvention encompasses compositions made according to any of the methodsfor preparing compositions disclosed herein.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Any embodiment, element, feature, application, or aspect ofthe compositions and/or methods of the invention can be excluded fromany one or more claims. For purposes of brevity, all of the embodimentsin which one or more elements, features, purposes, or aspects isexcluded are not set forth explicitly herein.

INCORPORATION OF REFERENCES

All publications and patent documents cited in this application areincorporated by reference in their entirety to the same extent as if thecontents of each individual publication or patent document wereincorporated herein.

1-65. (canceled)
 66. A pharmaceutical composition formulated fortreating hyperphosphatemia comprising a therapeutically effective amountof chitosan, wherein the chitosan has a degree of de-acetylation greaterthan about 95% and is characterized with an ability to bind at leastabout 60 mg phosphate per gram of chitosan in an in vitrophosphate-binding assay, and wherein the therapeutically effectiveamount of chitosan ranges from greater than 3.0 grams to about 20 gramsper dose.
 67. The pharmaceutical composition of claim 66, wherein thehyperphosphatemia is associated with chronic kidney disease and/orend-stage renal disease.
 68. The pharmaceutical composition of claim 66,wherein the hyperphosphatemia is associated with one or more disordersof phosphate metabolism and/or impaired phosphate transport function.69. The pharmaceutical composition of claim 66, wherein thetherapeutically effective amount ranges from greater than 3.0 to about10 grams chitosan per dose.
 70. The pharmaceutical composition of claim66, wherein the chitosan is characterized with the ability to bind atleast about 90 mg phosphate per gram in the in vitro phosphate-bindingassay.
 71. The pharmaceutical composition of claim 66, wherein thechitosan is characterized with the ability to bind at least about 120 mgphosphate per gram in the in vitro phosphate-binding assay.
 72. Thepharmaceutical composition of claim 66, wherein the chitosan ischaracterized with the ability to bind at least about 150 mg phosphateper gram in the in vitro phosphate-binding assay.
 73. The pharmaceuticalcomposition of claim 66, wherein the chitosan is characterized with theability to bind at least about 180 mg phosphate per gram in the in vitrophosphate-binding assay.
 74. The pharmaceutical composition of claim 66,wherein the chitosan is present in a form of a plurality of particles.75. The pharmaceutical composition of claim 74, wherein the plurality ofparticles have a mean volume particle size less than about 100 cubicmicrons.
 76. The pharmaceutical composition of claim 74, wherein theplurality of particles have a median volume particle size less thanabout 100 cubic microns.
 77. The pharmaceutical composition of claim 74,wherein the plurality of particles comprise one or more particles havinga roundness greater than about
 10. 78. The pharmaceutical composition ofclaim 77, wherein at least about 0.3% of the plurality of particles havea roundness greater than
 10. 79. The pharmaceutical composition of claim66, wherein the pharmaceutical composition is formulated for oraladministration.
 80. The pharmaceutical composition of claim 66, whereinthe pharmaceutical composition is a nutritional supplement.
 81. Thepharmaceutical composition of claim 66, wherein the pharmaceuticalcomposition further comprises a carrier.
 82. The pharmaceuticalcomposition of claim 81, wherein the carrier is selected from the groupconsisting of a starch, a gum, an alginate, a silicate, dextrose,gelatin, lactose, mannitol, sorbitol, sucrose, tragacanth, cellulose,methyl cellulose, microcrystalline cellulose, a methylhydroxybenzoate, apropylhydroxybenzoate, polyvinylpyrrolidone and talc.
 83. Thepharmaceutical composition of claim 66, wherein the pharmaceuticalcomposition is in a form of a cachet, a hard gelatin capsule, a softgelatin capsule, an elixir, a lozenge, a pill, a powder, a sachet, asterile packaged powder, a suspension, a syrup, or a tablet.
 84. Anutritional supplement comprising chitosan, wherein the chitosan has adegree of de-acetylation greater than about 95% and is characterizedwith an ability to bind at least about 60 mg phosphate per gram ofchitosan in an in vitro phosphate-binding assay, and wherein thechitosan is present in a form of a plurality of particles having a meanvolume particle size less than about 100 cubic microns.
 85. Thenutritional supplement of claim 84, wherein the plurality of particlescomprise one or more particles having a roundness greater than about 10.86. The nutritional supplement of claim 84, wherein at least 0.3% of theplurality of particles have a roundness greater than about
 10. 87. Anutritional supplement comprising chitosan, wherein the chitosan has adegree of de-acetylation greater than about 95% and is characterizedwith an ability to bind at least about 60 mg phosphate per gram ofchitosan in an in vitro phosphate-binding assay, and wherein thechitosan is present in a form of a plurality of particles having amedian volume particle size less than about 100 cubic microns.
 88. Thenutritional supplement of claim 87, wherein the plurality of particleshave a mean volume particle size less than about 100 cubic microns. 89.The nutritional supplement of claim 87, wherein the plurality ofparticles comprise one or more particles having a roundness greater thanabout
 10. 90. The nutritional supplement of claim 87, wherein at least0.3% of the plurality of particles have a roundness greater than about10.